Tyrosine Decarboxylase and Dopa Decarboxylase in Drosophila virilis Under Normal Conditions and Heat Stress: Genetic and Physiological Aspects

The activity of tyrosine decarboxylase (TDC) and dopa decarboxylase (DDC) was studied in adults of two lines of Drosophila virilis,contrasting in their reaction to stress conditions. Differences were found in the activity of both enzymes between individuals of the examined lines. Genetic analysis of these differences was made. Each of the two enzymes was found to be controlled by a single gene or, possibly, by a block of closely linked genes. The gene responsible for TDC activity is located on one of the autosomes (excluding chromosome II). DDC activity in D. virilisis regulated by a gene located, apparently, on chromosome II. Adults of the line responding to stress by a stress reaction (r-line) were shown to react to a short-term heat stress (38°C, 60 min) by a decrease in TDC activity. TDC activity in flies of the line incapable of the stress reaction (nr-line) did not alter in such conditions. DDC activity of adults of both lines was found to be unchangeable under stress conditions.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases ^1-7. Progression of tumors from a benign to a metastatic state is a complex event ⁸ and has been modeled in Drosophila to help us better understand the genetic basis of this disease ⁹. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system ¹⁰ and exploits the cooperation between an activated oncogene, Ras^V12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors ⁹. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.     

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.     

Drosophila female germline stem cells undergo mitosis without nuclear breakdown

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Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile⁵⁰⁸, Asn⁵⁰⁹, and Asp⁵¹¹) in communicating with converter domain residue Arg⁷⁵⁹. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.     

The persistence of short‐term cold acclimation in Drosophila melanogaster (Diptera: Drosophilidae)

Daily and seasonal fluctuations in temperature present significant challenges for the survival of many ectothermic species that can be tempered via thermal acclimation. In the present study, we use multiple naturally derived genotypes of Drosophila melanogaster to determine the persistence of beneficial short‐term thermal acclimation on subsequent survival after cold shock. We found that the benefit of short‐term acclimation persisted for 2 h in most genotypes after a rapid cold hardening treatment. Genotype did not directly influence the persistence of short‐term acclimation benefits, indicating that environmental variation may be more important for the persistence of acclimation benefits rather than genetic capacity for acclimation. The present study extends the current understanding of the limits and importance of short‐term acclimation events, providing greater detail on the timing of the loss of short‐term acclimation benefits in a genetically variable natural population.